It is estimated that 7?10% of the general population suffers from chronic neuropathic pain and associated annual economic costs exceed $160 billion in the US. Current analgesics used to treat chronic neuropathic pain lack efficacy, induce dose-limiting side effects or present a significant risk of tolerance and abuse. Thus, the discovery of novel analgesics that provide meaningful pain relief with improved safety, tolerability and reduced abuse potential remains an unmet medical need. Among the various mechanisms involved in pathological pain, disinhibition of nociceptive signaling from the spinal cord to the higher CNS plays a critical role. Indeed, we and others have shown that inhibitory glycinergic neurotransmission in the dorsal horn is impaired in pathological pain states. Thus, it has been proposed that enhancing spinal glycinergic neurotransmission could reduce nociceptor signaling and provide analgesia. Glycine transporter-2 (GlyT-2) regulates extracellular glycine concentrations in the CNS and presents a highly attractive target to augment impaired spinal inhibitory signaling. Indeed, GlyT-2 inhibitors have demonstrated efficacy in several rodent models of acute and chronic pain. Recent studies suggest that either partial or reversible GlyT-2 inhibition can potentially circumvent potential mechanism-based adverse effects and provide analgesics with a suitable balance of efficacy and tolerability. We synthesized 60 bioactive lipid allosteric partial inhibitors exhibiting varying degrees of potency and %maximal inhibition and our most potent compound of this class enhances tonic glycinergic currents ex vivo in rat spinal cord slices without depleting presynaptic glycine reloading and produces in vivo dose-dependent efficacy in PNL rats without adverse effects. Separately, we synthesized 18 novel GlyT-2 inhibitor hit compounds derived from ORG-25543 that exhibit reproducible variations in reversibility and transport recovery using a washout assay protocol. From these two libraries, we propose conducting a dual-pronged medicinal chemistry campaign to identify optimized compounds possessing favorable potency, binding profiles and ADME characteristics suitable for measuring effects in spinal cord slices and in vivo PK. Selected compounds will be screened in the rat PNL model to assess analgesic efficacy and in behavioral models for dose-limiting effects and TI. Our proposal seeks support to optimize our hit compounds to provide selective and orally bioavailable GlyT-2 inhibitors for further development and eventual human clinical trials in neuropathic pain. The studies outlined in this proposal seek to conduct medicinal chemistry optimization using a battery of primary, secondary, counter-screen, and ADMET assays to assess potency, binding profile (%maximal inhibition, reversibility and mechanism of binding), selectivity and drug- like properties (Specific Aim 1), and assess PK, preclinical analgesic efficacy and tolerability of advanced compounds in animal models of neuropathic pain and motor behavior at different stages of compound optimization (Specific Aim 2).